Attenuated smallpox vaccine strain

ABSTRACT

The present invention discloses an attenuated smallpox vaccine strain exhibiting antibody production similar to conventional strains but without postvaccinal side effects. The vaccine is prepared by attenuating a Lister strain of a vaccinia virus by cell culture and selecting a suitable strain therefrom showing relatively small and uniform pocks on the chorioallantoic membrane of an embryonated egg.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a vaccine strain exhibiting a similar antibody productivity to that of conventional strains and little postvaccinal side effects, which is prepared by attenuating a Lister strain of a vaccinia virus by cell culture.

(1) Preparation of the Strain of the Present Invention

The original strain (i.e. Lister strain) was subcultured in rabbit Kidney cells over 36 generations at 30° C. and then plaque-purified thrice to isolate 50 clones. From these 50 clones, a temperature-sensitive variant which showed the worst growth at 40° C. in Vero cells established from green monkey (Coropithecus aethiops) Kidney cells was selected. Compared with the original strain, the temperature-sensitive variant grew better in rabbit Kidney cells but worse in rabbit central nervous system cells. In the central nervous system of a monkey, it exhibited a pathogenicity which was similar to that of DI_(s) strain, i.e. an attenuated micropock variant prepared from Dairen I strain, and extremely lower than that of the original strain. Accordingly an inoculation test was carried out with the use of a small number of subjects. Consequently it was found that the foregoing variant would result in a light systemic reaction with a febrility ratio of 14% followed by somewhat slow formation of crust. Therefore it was attempted to isolate a clone showing a poor skin growth capacity. As a marker in the isolation of the foregoing clone, the size of pocks on the chorioallantoic membrane of an embryonated egg was employed. That is, the temperature-sensitive variant was subcultured in rabbit Kidney cells over six generations and plaque-purified twice to isolate a clone showing relatively small and uniform pocks on the chorioallantoic membrane of an embryonated egg. The clone was further subcultured in rabbit Kidney cells over additional three generations at 30° C. to isolate a clone showing extremely small pocks on the chorioallantoic membrane of an embryonated egg to thereby prepare an attenuated smallpox vaccine strain.

(2) Properties of the Strain of the Present Invention

Properties of the strain of the present invention are summarized in Table 1 compared with those of the original strain (i.e. Lister strain).

As shown in Table 1, the strain of the present invention has many markers available in tests in vitro and exhibit a lower pathogenicity in the central nervous systems of rabbits and cynomolgus monkeys (Macaca fascicularis), no invasiveness caused by peripheral infection on the central nervous system of mice and a poor skin growth capacity in rabbits and human. These properties suggest that it is available as a smallpox vaccine strain.

                  TABLE 1                                                          ______________________________________                                         Properties of Lister strain and the strain of                                  the present invention                                                                          Lister    Strain of                                                            strain    the invention                                        ______________________________________                                         Ingrowable temperature                                                                           41° C. or above                                                                     40.5° C.                                  in rabbit Kidney cells                                                         Plaque size in rabbit                                                                            large       medium                                           Kidney cells                                                                   Pock size         large       medium                                           Growability in chorio-allantoic                                                                  +++         +++ *1                                           membrane of an                                                                 embryonated egg                                                                Growability in Vero cells                                                                        +++           + *1                                           Pathogenicity in central                                                       nervous system                                                                 Rabbit *2         +++         +                                                Cynomolgus monkey *3                                                                             yes (died)  no (survival)                                    Invasiveness by peripheral                                                     infection of central nervous                                                   system *4                                                                      Mouse (cortisone acetate,                                                                        yes         no                                               subcutaneous inoculation)                                                      Mouse (untreated) yes         no                                               Skin growth capacity                                                           Rabbit            +++         +                                                Human              ++         +                                                ______________________________________                                          *1 They differ from each other by 2log.sub.10.                                 *2 Evaluated from the amount of recovered virus six days after                 intracerebral inoculation of 10.sup.6.7 TCID.sub.50 of a virus.                *3 Evaluated by intrathalmic inoculation of 10.sup.8.5 TCID.sub.50 of a        virus.                                                                         *4 Mice treated or untreated with cortisone acetate were intraabdominally      inoculated with 10.sup.7.3 PFU of a virus and the amounts of intracerebra      virus thereof were determined.                                                 Results are shown in FIGS. 1 and 2.                                      

The antibody productivity of the strain of the present invention in a rabbit was compared with that of the Lister strain. Table 2 shows the results. In spite of the poor skin growth capacity, the strain of the present invention brought about similar increase and continuance in both hemagglutination inhibitting antibody titer and neutralizing antibody titer to those of the Lister strain.

                  TABLE 2                                                          ______________________________________                                         Change in antibody titers                                                               Hemagglutination                                                               inhibitting      Neutralizing                                                  antibody titer   antibody titer                                       Weeks after                                                                               Lister  Strain of  Lister                                                                               Strain of                                  inoculation                                                                               strain  invention  strain                                                                               invention                                  ______________________________________                                         2          2.sup.6.5                                                                              2.sup.4.5  4.sup.4.9                                                                            4.sup.4.9                                  4          2.sup.6.5                                                                              2.sup.5.0  4.sup.4.9                                                                            4.sup.4.7                                  6          2.sup.4.5                                                                              2.sup.5.0  4.sup.5.0                                                                            4.sup.4.6                                  13         2.sup.3.5                                                                              2.sup.5.5  4.sup.5.0                                                                            4.sup.4.7                                  ______________________________________                                          Note: Each strain was inoculated in an amount of 10.sup.8.0 PFU.         

(3) Determination of Infectivity Titer

Vero cells which have been generally used in determining infectivity titers of vaccinia viruses are unsuitable in determining the infectivity titer of the strain of the present invention because of the poor sensitivity thereto. On the other hand, the pock method with the use of the chorioallantoic membrane of an embryonated egg is highly sensitive to the strain of the present invention although it has been pointed out that various factors such as the viral dilution, variation between embryonated eggs and variation in the techniques of workers would result in a poor reproducibility of this method. The plaque method with the use of chick embryo cells which is employed herein is somewhat less sensitive than the pock method, but results in infectivity titers of a high reproducibility. Therefore the latter is an effective method in determining the infectivity titer of the strain of the present invention. The result of an examination on the correlativity and reliability of the pock and plaque methods is as follows.

                  TABLE 3                                                          ______________________________________                                         Comparison of infectivity titers determined by                                 pock and plaque methods                                                                          Pock      Plaque                                             Smallpox vaccine strain                                                                          PFU/ml    PFU/ml                                             ______________________________________                                         Lister strain     1.19 × 10.sup.8                                                                    1.38 × 10.sup.7                                "               4.97 × 10.sup.7                                                                    1.43 × 10.sup.7                              Strain of the invention                                                                          2.77 × 10.sup.8                                                                    5.56 × 10.sup.7                                "               1.47 × 10.sup.8                                                                    2.90 × 10.sup.7                                "               1.33 × 10.sup.8                                                                    5.69 × 10.sup.7                                "               1.13 × 10.sup.8                                                                    3.0  × 10.sup.7                                "               1.51 × 10.sup.6                                                                    5.49 × 10.sup.5                              DI.sub.s strain   1.44 × 10.sup.6                                                                    2.00 × 10.sup.5                                "               9.77 × 10.sup.5                                                                    2.27 × 10.sup.5                              ______________________________________                                    

Table 3 shows the infectivity titers of the Lister strain of vaccinia virus, the strain of the present invention and a DI_(s) strain of the same virus simultaneously determined by these two methods. The correlation coefficient γ calculated by logarithmic infectivity titers determined by these two methods is 0.984. As a result of a test of significance of the correlation, t₀ is determined to be 14.52 which is larger than t_(f=7) =(α=0.001)=54.08, suggesting that the infectivity titers determined by these two methods closely relate to each other. The regression equation of these infectivity titers determined by these two methods is y₁ =0.838+0.971y₂, wherein y₁ (log₁₀ TCID₅₀ /ml) represents infectivity titers determined by the pock method while y₂ (log₁₀ TCID₅₀ /ml) represents those determined by the plaque method.

In order to examine the reliability of these two methods, variance of the data obtained by these methods were compared with each other. Table 4 (A) shows the data obtained by measuring solutions prepared by appropriately diluting the same strains as used in Table 3 by these two methods. Variance and variant ratio of these data of each virus were determined for F calibraliton to thereby test the significance of the variance between these two methods. Results is shown in Table 4 (B).

                                      TABLE 4                                      __________________________________________________________________________     Data obtained by pock and plaque methods and variant ratio thereof                    A             B                                                                pock   plaque                                                                  method method variance                                                  Smallpox                                                                              number number pock                                                                               plaque                                                                             variant                                           vaccine                                                                               of pocks                                                                              of plaques                                                                            method                                                                             method                                                                             ratio  F                                          strain /0.1 ml                                                                               /0.1 ml                                                                               (S.sub.1.sup.2)                                                                    (S.sub.2.sup.2)                                                                    F.sub.0 = S.sub.1.sup.2 /S.sub.2.sup.2                                                α = 0.05                             __________________________________________________________________________     Lister 31.34.35.38.39                                                                        24.24.27.28                                                                           278.2                                                                              11.41                                                                              24.38  f.sub.1 = 9 f.sub.2 = 7                    strain 50.57.68.70.75                                                                        31.31.32.32           3.677                                      Strain of                                                                             40.41.42.59                                                                           13.17.18.21.22                                                                        164.8                                                                              46.9                                                                                3.515 f.sub.1 = 7 f.sub.2 = 9                    the invention                                                                         61.62.63.75                                                                           22.23.23.25.39        3.293                                      Strain of                                                                             14.34.35.52.52                                                                        29.29.31.32.37                                                                        405.7                                                                              44.6                                                                                9.096 f.sub.1 = 8 f.sub.2 = 9                    the invention                                                                         52.62.73.78                                                                           37.38.40.43.50        3.230                                      DI.sub.s strain                                                                       26.29.37.44                                                                           19.20.20.22                                                                           387.8                                                                              12.57                                                                              30.85  f.sub.1 = 6 f.sub.2 = 6                           62.71.73                                                                              24.25.29              4.284                                      DI.sub.s strain                                                                       13.14.20.23.25                                                                        7. 7. 8. 8                                                                            146.2                                                                              1.0 146.2  f.sub.1 = 9 f.sub.2 = 6                           27.36.38.42.49                                                                        8. 8. 10              4.099                                      __________________________________________________________________________

Consequently F₀ is larger than F (α=0.05) in all viral samples. That is to say, there is a significant difference in the variance at a ratio of risk of 5%, which suggests that the pock method would bring about higher variant than the plaque method in determining the infectivity titer of a vaccinia virus. Accordingly these two methods closely correlate to each other and the latter is much more reliable than the former.

However, these two methods may be selected case by case. For example, it is preferable to employ the plaque method in comparing infectivity titers, while it is preferable to employ the pock methods in determining absolute infectivity titers.

EXAMPLE Preparation of Dried Smallpox Vaccine in Cell Culture

A conventional process for preparing a variola vaccine comprises inoculating bovine skin with a vaccinia virus and scratching a portion on which pocks are formed to thereby use it as a vaccine in the form of an emulsion. However, the vaccine thus prepared may be contaminated with various bacteria, mycoplasma and viruses other than the vaccinia virus. It is very difficult to prepare the vaccine not contaminated with any microorganism. On the contrary, the cell cultured vaccine prepared from the strain of the present invention may be contaminated with no microorganism. Adding to the ideal property from a viewpoint of quality control, the vaccine thus prepared is superior to conventional ones in postvaccinal side effects and complicating diseases. Furthermore the cell cultured vaccine prepared from the strain of the present invention is superior to conventional methods for vaccination with the use of the chorioallantoic membrane of an embryonated egg or bovine skin from an economical viewpoint and may be lyophilized by diluting with a medium and adding an appropriate stabilizer.

(4) Process for the Preparation

(1) Cell culture

A kidney of a rabbit aged three weeks or younger was removed, cut into pieces under a sterile condition, digested with a 0.25% solution of trypsin and suspended in a medium for cell-growth containing 10% of Calf serum to give a concentration of 3×10 cells/ml. 200 ml portions of the medium were introduced into bottles and subjected to roller bottle culture at 37° C. for four days.

(2) Culture of virus

After removing the medium, the surface of the cells was washed with a saline solution twice and each roller culture bottle was inoculated with 10 ml (1.55×10⁷ PFU/ml) of the strain of the present invention followed by adsorption at 30° C. for two hours. Then 100 ml of a 199 medium containing 0.2% of gelatin was added and the mixture was cultured at 30° C. for two or three days. After shaking the cells off from the bottle, the cell suspension was centrifuged at 2,000 rpm for 10 min. After removing the supernatant, the cells were resuspended in 10 ml of a 199 medium containing 0.2% of gelatin.

(3) Extraction of virus

The cell suspension was ultrasonically treated and centrifuged at 2,000 rpm for 10 min. The supernatant thus separated was employed as the bulk material of the vaccine.

(4) Determination of infectivity titer

The chorioallantoic membrane of an embryonated egg aged 11 days was inoculated with the viral solutions and cultured at 37° C. for two days. Pocks thus formed were counted to culculate the infectivity titer per ml. The infectivity titer of the pool cell extract was 1.7×10⁸ PFU/ml.

(5) Lyophilization

The virus extracted from the cells was diluted with a 199 medium and sorbitol and peptone were added thereto to give a final concentration of 5% each. 0.7 ml portions of the obtained solution were introduced into vial bottles and lyophilized with a vacuum lyophilizer. Table 5 shows the result.

                  TABLE 5                                                          ______________________________________                                         Result of lyophilization                                                       Infectivity titer (PFU*/ml)                                                    before     after                 Moisture                                      lyophilization                                                                            lyophilization                                                                              lowering content                                       ______________________________________                                         1.7 × 10.sup.8                                                                      1.5 × 10.sup.8                                                                        1/1.2    0.9-1.8                                       ______________________________________                                          *pock forming units                                                      

(6) Shelf stability of lyophilized vaccine

The lyophilized vaccine was subjected to an accelerated denaturation test by storing at 30° C., 37° C., 50° C. and 60° C. thus estimating the decrease in the infectivity titer and comparing it with the minimum requirement of biological products as well as that of a test of the stability of cell cultured smallpox vaccine. Each sample was collected three or four times depending on the temperature at which it was stored. Infectivity titers of samples in two vial bottles were evaluated each time separately. Table 6 shows the result.

                                      TABLE 6                                      __________________________________________________________________________     Infectivity titer of samples stored at various                                 temperature vs. time                                                           Heating                                                                        period                                                                              Infectivity titer and decrease                                            __________________________________________________________________________     30° C.                                                                       0      2 weeks   4 weeks  23.6 weeks                                            5.40 × 10.sup.7 *                                                              2.73 × 10.sup.7 1/2.0**                                                            2.39 × 10.sup.7 1/2.3                                                             4.83 × 10.sup.6 1/11.2                                3.09 × 10.sup.7 1/1.7                                                              2.42 × 10.sup.7 1/2.2                                                             5.86 × 10.sup.6 1/9.2                     37° C.                                                                       0      1 week    4 weeks  7 weeks  23.6 weeks                                  5.40 × 10.sup.7                                                                 3.11 × 10.sup.7 1/2.1                                                              1.40 × 10.sup.7 1/3.9                                                             1.00 × 10.sup.7 1/5.1                                                             1.67 × 10.sup.6 1/32.4                       5.71 × 10.sup.7 1/0.9                                                              1.11 × 10.sup.7 1/4.9                                                             8.15 × 10.sup.6 1/6.6                                                             1.68 × 10.sup.6 1/32.0           50° C.                                                                       0      4 days    9 days   17 days  4 weeks                                     5.40 × 10.sup.7                                                                 1.09 × 10.sup.7 1/5.0                                                              5.75 × 10.sup.6 1/9.4                                                             1.72 × 10.sup.6 1/31.4                                                            1.11 × 10.sup.6 1/48.8                       7.79 × 10.sup.6 1/6.9                                                              4.05 × 10.sup.6 1/13.3                                                            4.75 × 10.sup.6 1/11.4                    60° C.                                                                       0      2 days    4 days   9 days   17 weeks                                    5.40 × 10.sup.7                                                                 2.31 × 10.sup.6 1/23.4                                                             3.30 × 10.sup.6 1/16.4                                                            3.26 × 10.sup.5 1/16.6                                                            1.13 × 10.sup.5 1/477                        3.05 × 10.sup.6 1/17.7                                                             9.93 × 10.sup.5 1/54.3                                                            2.05 × 10.sup.5 1/26.4                                                            7.98 × 10.sup.3                  __________________________________________________________________________                                             1/6723                                  *plaque forming units (PFU)/ml                                                 **decrease in the infectivity titer based on that at the initiation.     

The residual infectivity titer of the sample stored at 37° C. for four weeks was 1/44 and higher than the minimum requirement of biological products (1/10). The infectivity titer thereof was 10⁷.8 PFU/ml and also higher than the minimum requirement of biological products (10⁷.7 PFU/ml). Therefore both of these two values satisfy the minimum requirement of biological products.

Decreases in the infectivity titers at these temperatures were calculated according to the equation of an accelerated denaturation test based on the data as shown in Table 6. Table 7 shows the result.

                  TABLE 7                                                          ______________________________________                                         Estimated shelf stability by accelerated                                       denaturation test                                                              Presumed temp.                                                                 for storage                                                                              20%-decrease period                                                                            Half-value period                                    ______________________________________                                         -70° C.                                                                           6.2 × 10.sup.5 ˜ 7.6 ×                                                       1.9 × 10.sup.6 ˜ 2.4 ×                       10.sup.8 years* 10.sup.9 years*                                      -15°                                                                              14.8 ˜ 266 years                                                                         45.9 ˜ 828 years                                 5°                                                                              320 days ˜ 5.1 years                                                                     2.7 ˜ 160 years                                 15°                                                                              90.1 ˜ 321 days                                                                          280 days ˜ 2.7 years                            25°                                                                              27.7 ˜ 61.7 days                                                                         86 ˜ 190 days                                   37°                                                                              7.4 ˜ 9.8 days                                                                           23.1 ˜ 30.5 days                               ______________________________________                                          *a confidence interval of 95%.                                           

The 20%-decrease period and half-value period of the sample stored at -15° C. were longer than 14 years and 45 years respectively, which suggests that the vaccine may be stored for a long time.

(5) Field Test of the Strain of the Present Invention

In order to examine the presence of postvaccinal side effects and complicating diseases caused by the strain of the present invention based on the antibody productivity thereof, a field test was carried out with the guidance of the Ministry of Health and Welfare of Japan. The result will now be summarized.

(1) Antibody production

The antibody productivity of the strain of the present invention was compared with those to the Lister strain and the CV-1 strain which has been subcultured in chick embryo cells at New York City Department of Health Laboratory. Table 8 shows the result.

                                      TABLE 8                                      __________________________________________________________________________     Antibody production after inoculation of various                               smallpox vaccine strains                                                       Smallpox vaccine                                                                         Time Number of                                                       strain    (month)                                                                             subjects                                                        __________________________________________________________________________                          HI antibody titer                                                              <2 ˜ 4                                                                        2  4  8   16  32 64 128 average                      Strain of invention                                                                      1 ˜ 11/2                                                                      513   18   12 88 161 155 72 6  1   2.sup.3.3                              3 ˜ 6                                                                         19    1    3  3  2   2   6         2.sup.3.2                    CV-1      1 ˜ 11/2                                                                      26            1  7   11  7         2.sup.3.9                              3 ˜ 6                                                                         25            2  10  11  1  1      2.sup.3.6                    Lister    1 ˜ 11/2                                                                       7                   5   2         2.sup.4.3                              3 ˜ 6                                                                         19            4  5   8   2         2.sup.3.4                    __________________________________________________________________________                          NT antibody titer                                                              <4.sup.0.9                                                                          4.sup.1.0˜                                                                  4.sup. 1.5˜                                                                 4.sup.2.0˜                                                                   4.sup.2.5˜                                                                   4.sup.3.0˜                                                                  4.sup.3.5˜                                                                  4.sup.4.0˜                                                                   average                      Strain of invention                                                                      1 ˜ 11/2                                                                      97         5  10 25  37  18 2      4.sup.2.5                    CV-1      1 ˜ 11/2                                                                       6    1       2  2       1         4.sup.1.9                              3 ˜ 6                                                                         11    1    1  4  2   3             4.sup.2.0                    Lister    1 ˜ 11/2                                                                       5               3   2             4.sup.2.4                              3 ˜ 6                                                                         12    1    2     2   7             4.sup.2.2                    __________________________________________________________________________

As shown in Table 8, both of the hemagglutination inhibitting (HI) and neutralizing (NT) antibody titers suggest that the strain of the present invention would have a similar or superior antibody productivity to those of the Lister and CV-1 strains. As a result of a booster with the Lister strain to 561 subjects previously inoculated with the strain of the present invention and an immunological examination on their topical reacitons, it was found that sufficient immunity would be obtained by the inoculation with the strain of the present invention.

(2) Abnormality in brain waves

A brain wave test was performed on those showing positive reactions two weeks after the inoculation with the strain of the present invention or the Lister strain. Consequently five subjects among 19 (26.3%) inoculated with the Lister strain exhibited temporary abnormality in brain waves while those inoculated with the strain of the present invention exhibited no abnormality, which suggests that the latter strain has a low neurotropsy.

(3) Postvaccinal side effects and complicating diseases

Approximately fifty thousands children have been inoculated with the strain of the present invention without any serious side effects nor complicating diseases. 10,578 children among them were clinically followed in detail after the inoculation. Tables 9 and 10 show the ratios of positive reaction by age, febrility and complicating diseases.

                                      TABLE 9                                      __________________________________________________________________________     Ratio of positive reaction and febrility caused by                             the strain of the present invention by age                                     __________________________________________________________________________                       Those showing positive reaction and examined systemic                          reaction for 14 days or longer                                                       Number of                                                        Number of the subjects                                                         subjects      attacked                                                         showing       with fever                                                                            Basal temperature                                   Number of                                                                            positive                                                                               Number of                                                                            after 4 to            un-                              Age subjects                                                                             reaction                                                                               subjects                                                                             14 days                                                                               37.5˜                                                                       38.0˜                                                                        39.0˜                                                                        40.0˜                                                                        known                            __________________________________________________________________________     0     11    11      11   0      0  0   0  0   0                                1     687   669     632  69     14                                                                                42 11  0   2                                2 ˜ 4                                                                        6,800 6,496   6,094 474    139                                                                               219 88  6   22                               >5  2,040 1,900   1,807 120     42                                                                                57 17  3   1                                Total                                                                              9,538 9,075   8,544 663    195                                                                               318 116 9   25                                         (95.2%)       (7.8%) (29.4)                                                                            (48.0)                                                                             (17.5)                                                                             (1.4)                                                                              (3.8)                            __________________________________________________________________________     Those showing positive reaction                                                and examined systemic                                                           reaction for 14 days or longer                                                                        Other reactions                                        Febrility period              Auto-                                                                              Secondary                                                        un- Eczema                                                                               inocu-                                                                             vacciniae   Heat                             Age 1 day                                                                              2 days                                                                             3 days                                                                             4 days                                                                             known                                                                              vaccinatum                                                                           lation                                                                             vesicle                                                                              exanthema                                                                            cramps                           __________________________________________________________________________     1    33 19  8   8   1   1     2   1     3     1                                2 ˜ 4                                                                        288 115 38  25  8         6   25    5     2                                >5   81 28  5   6             1   2                                            Total                                                                              402 162 51  39  9   1     9   28    8     3                                    (60.6)                                                                             (24.4)                                                                             (7.7)                                                                              (5.9)                                                                              (1.4)                                                      __________________________________________________________________________

                  TABLE 10                                                         ______________________________________                                         Comparison of reactions caused by various smallpox                             vaccine strains                                                                                         Average                                                                               Average                                        Variola                                                                               Number   Ratio of rubor  induration                                                                             Febrility                              vaccine                                                                               of       positive size   size    ratio                                  strain subjects reaction (mm)   (mm)    (%)                                    ______________________________________                                         Lister  3,662   93.7     17.6   15.3    26.6                                   strain                                                                         CV-1   22,976   92.4     21.1   16.8    8.5                                    Strain of                                                                             10,578   95.1     18.4    6.1    7.7                                    the                                                                            invention                                                                      ______________________________________                                    

The ratio of positive reaction, the febrility ratio and the incidence of complicating skin diseases caused by the strain of the present invention were 95.2%, 7.8% and 0.19%, respectively. No complicating disease was observed in the central nervous system. The ratio of positive reaction caused by the strain of the present invention is similar to those caused by conventional smallpox vaccine strains, while the febrility ratio of the former is significantly lower than the latter. It might be further assumed that the incidences of postvaccinal encephalopathy and encephalitis caused by the former would be lower than those caused by the latter although the number of examples was not so large. Furthermore the strain of the present invention was superior in the average induration size as shown in Table 10.

Apart from the abovementioned purpose for preventing smallpox by the inoculation of a vaccinia virus, the strain of the present invention is further available in a recombinant DNA experiment wherein a recombination vaccinia virus containing foreign proteins is constructed by inserting DNA of an expressed polypeptide into the DNA of the vaccinia virus.

Since the strain of the present invention would exhibit no invasiveness caused by peripheral infection on the central nervous system of a mouse, little postvaccinal side effects and no postvaccinal encephalopathy nor encephalitis, it may be very advantageously employed in constructing the abovementioned recombination vaccinia virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the recovery of the virus from the blood and brain of a mouse inoculated with the Lister strain. FIG. 2 is a diagram of the recovery of the virus from the blood and brain of a mouse inoculated with the strain of the present invention. 

We claim:
 1. An attenuated smallpox vaccine strain exhibiting no invasiveness caused by peripheral infection on the central nervous system of a mouse and little post-vaccinal side effects, which is prepared by subculturing a Lister strain of vaccinia virus in rabbit kidney cells over 36 generations at 30° C., plaque-purifying the strain thrice to isolate 50 clones, selecting a temperature-sensitive variant showing worst growth in Vero cells at 40° C. from said 50 clones, subculturing the temperature sensitive variant in rabbit kidney cells over six generations, plaque-purifying the variant twice to isolate a clone showing relatively small and uniform pocks on the chorioallantoic membrane of an embryonated egg, subculturing the clone in rabbit kidney cells over three generations at 30° C. and isolating a clone showing very small pocks on the chorioallantoic membrane of an embryonated egg. 